Evidence suggests that semen has been collected for artificial insemination (AI) since the 1300's. It is held that Arabian tribes stole semen from rival tribes' stallions to inseminate their own mares and that the semen from a poor stallion was also used to inseminate the rival tribes' mares. The first documented use of AI was in the 1780's by the Italian physiologist, Spallanzani. His insemination of a bitch, with freshly collected semen, resulted in the production of three puppies. AI has continued to be developed as a tool in animal and human reproduction. Initially, a major problem with AI was that the collected semen had to be used the same day (and in cases such as with dogs, almost immediately) in order to achieve good results (i.e., pregnancy). In order for AI to develop to its full potential, a method had to be discovered to preserve semen for use at a later date.
The first successful, and by far still the most common, way to preserve semen for later use is with semen extenders. Semen extenders are used to provide nutrients for sperm metabolism, to carry additives such as antibiotics and cryoprotectants (for storage at lower temperatures), and to provide multiple breedings from one semen sample. In the 1930's, it was discovered that it was possible to use a buffered nutrient medium to extend the fertilizing life of semen for periods of up to three or four days. Extended semen can be maintained for a considerable length of time (times vary depending on the species) if the semen is chilled. Further, when a cryopreservative such as glycerol is added, extended semen can also be frozen and can remain viable (i.e., produce a pregnancy) for up to 20 years as shown in bovine semen.
Semen extenders have traditionally been added post-collection. After the semen is collected, the extender is normally added at a 1:1, 2:1, or 3:1 extender to semen ratio, depending on the initial motility, concentration, and species collected. In practice, the extender is added anywhere from a few minutes post-collection, up to approximately one-half to one hour post-collection.
Unprotected, freshly ejaculated semen loses motility and therefore fertilizing capability rapidly, rendering it virtually useless in a matter of a few minutes to a few hours (time varies depending on species). In some species, such as the canine, zero percent motility can be reached in less than one hour. This loss of fertilizing capability is even a problem with extended semen. If the extended semen is chilled, the reduction in motility and other semen parameters can be slowed, but not eliminated. The loss of motility and fertilizing capability is especially a problem for frozen-thawed semen. It is generally accepted that fifty percent or more of the initial motility will be lost during the freezing and thawing process.
From its earliest stages as a spermatogonia to its final maturation, which, from a biochemical standpoint, does not normally occur until after it has entered the female tract, the spermatozoon needs a constant supply of nutrients to maintain its metabolic activity and to undergo the processes necessary for fertilization of the ova. As spermatogonia develop into spermatozoa in the seminiferous tubules, their nutrient needs are provided by the Sertoli cells, also called nurse cells. These so-called “nurse cells” secrete fluids containing proteins necessary for the sperm cells to grow and to mature into the spermatozoa. They also supply some of the energy requirements of the sperm through the production of lactate, which is converted to pyruvate by the spermatid mitochondria.
As the spermatozoa are released into the lumen of the seminiferous tubules and continue their journey through the rete testes to the epididymis, they continue to be bathed in fluid rich in proteins, energy substrates such as glucose and lactate, and a variety of other substances. When the sperm cells reach the epididymis, they are still immotile. In the cauda epididymis, the sperm are concentrated and stored in a highly favorable environment. The epididymal cells secrete fluid that is low in pH and has a high potassium-to-sodium ratio. These conditions allow the sperm to be stored and matured and still remain viable for an extended period of time.
When the sperm are ready to be ejaculated, they are forced into the vas deferens and on into the urethra and then out of the body. Once the sperm reach the urethra the concentrated fluid in which they are contained is mixed with seminal fluid secreted by the accessory sex glands. The secretions of these glands contain buffers, nutrients, and a variety of other organic and inorganic substances. The buffers such as phosphates and carbonate buffers are essential for protection against pH shifts as the semen is deposited into the hostile environment of the female tract. In most species, the site of semen deposition, the vaginal vault, is extremely acidic. Further, as the sperm continue to metabolize, waste products, such as lactic acid, are produced which can lower the pH even more. The organic and inorganic ions such as sodium (Na2+), potassium (K+), and calcium (Ca2+) are necessary to initiate sperm motility and fertilizing capability. Other nutrients, such as fructose and sorbitol, are utilized by the sperm to meet energy requirements. Extenders were created in an attempt to hold the sperm in a favorable environment for cellular survival, while biochemically placing the cells in suspended animation (delaying their progression toward final maturation) until time for their use.
Since Spallanzani's first documented use of AI in the 1780's, artificial insemination has continued to develop its niche in reproduction. Because the raw (unprocessed) semen lost fertilizing capability rapidly after ejaculation, Spallanzani discovered that the semen had to be used soon after collection in order to achieve good results. It would be one hundred and fifty years before a method would be developed to extend the fertilizing life of spermatozoa after ejaculation.
With the development of semen extenders, semen could be preserved for use many hours, or even days, post-ejaculation. First described in the 1930's, it was discovered that it was possible to use a buffered nutrient medium to extend the fertilizing life of semen for up to three or four days. Extenders are used in an attempt to hold sperm in optimal conditions until their use and also allow for multiple breedings from one semen sample.
In order for a semen extender to be effective, it must contain a number of ingredients. It must buffer the semen against shifts in pH due to the continual metabolic activity of the sperm during storage. It must also maintain an isotonic environment. If the extender is hypertonic, the sperm will shrivel and die. If the solution is hypotonic, the sperm will swell and burst. A number of ingredients have been used to meet these requirements. The first successful buffer to be used in a semen extender was the phosphate buffer. Although the phosphate buffer worked, the sodium citrate buffer soon replaced it, because when mixed with egg yolk (a common nutrient in semen extenders) the mixture remained transparent. A variety of other buffers and various combinations of these buffers are now available. Some of these buffers are the tris buffer solution, the tes buffer, the test-yolk buffer (combined tes and tris buffers with egg yolk), and tris-citrate buffer. Regardless of which buffer is being used, all are added in the proper concentrations to not only buffer the solution, but also to maintain the isotonic nature of the extender.
Semen extenders must contain adequate nutrients for sperm to metabolize during storage. A variety of substances fill this need. Milk and egg yolk are common protein sources. A third protein source is irradiated bovine albumin.
Simple sugars are added to provide the sperm with energy. Fructose, glucose, sucrose, sorbitol and pyruvate have all been used in semen extenders as sources of energy. Pyruvate, by its chemical nature, is the energy substrate most easily utilized by the sperm. However, it is not the most common energy source. The most common energy substrate found in commercial semen extenders is fructose. Its ability to easily be converted to pyruvate within the sperm mitochondria and its cost effectiveness make it an ideal source of energy.
In addition to their roles as nutrient sources, proteins also help fulfill another requirement of the semen extender by serving as cryoprotectants. Unprotected, the membranes of the spermatozoa undergo configurational changes as the temperature is lowered (cold shock). However, the lecithin, lipoproteins, and phospholipids from the protein source provide protection from cold shock. As the semen is lowered from body temperature to 5° C., the temperature at which fresh chilled semen is held during storage, the sperm will undergo cold shock if not protected. When properly prepared, chilling to this temperature keeps the semen viable for a longer period of time than if kept at approximately room temperature (15-20° C.). Further, non-protein cryoprotective agents must be employed if the semen are to be frozen. This will be discussed in detail below.
The final ingredient in most semen extenders is an anti-microbial agent. The anti-microbial agent is essential for reducing microbial contamination and preventing the spread of diseases that can be transported in the semen. Through their control of venereal diseases, these agents have also been shown to improve conception rates. Some common antibacterial agents are penicillin, streptomycin, lincomycin, and gentamicin.
When an extender is to be used as the base media for freezing and long-term storage of semen (cryopreservation), an additional cryoprotective agent must be added to protect spermatozoa from ice crystal formation. When semen is frozen without a cryoprotectant, the ice crystals that form during the freezing process puncture the cell membrane and result in cell death. By using a cryoprotectant such as glycerol or DMSO (dimethylsulphoxide), a large portion of the intracellular water is displaced by the cryoprotectant; therefore, cellular damage due to ice crystal formation is largely prevented.
Semen extenders have continued to be used since their inception and a variety of extenders and cryoprotectants are now available commercially. These products fall into three basic classes based on their protein source (egg yolk, milk, or albumin). The egg yolk based extenders are used in many species, including cattle, sheep, dogs, and humans. Milk based extenders are used almost exclusively in the horse. The third class of extenders, serum albumin based extenders, are used in species such as dogs, exotics and humans.
Artificial insemination has been used for several centuries. However, it was not until the early 1900's, when semen extenders were developed, that semen could be stored and used at a time other than immediately after collection. The traditional method of extending semen was, and still is, to add the extender anywhere from a few minutes up to one hour post-collection. While this method does help preserve semen for use at a later time post-collection, data from this experiment suggests it is not the most efficient method.
Semen collection can be performed in a variety of ways depending on the species. Methods include the artificial vagina (AV), electro-ejaculation, digital manipulation and masturbation.
The first artificial vagina was developed at the University of Rome in 1914 for use in the dog. Russian scientists developed other AV's for use in larger species such as the horse in 1933. Today's AV's, which vary in size and shape depending on species, consist of a tapered collection sleeve, in which the penis is placed. Attached to the tapered end is a collection container. Most AV's attempt to simulate natural copulation by providing suitable temperature, pressure and lubrication to induce ejaculation.
The electro-ejaculator was developed in the 1940's. It consists of a probe that is placed into the rectum of the male to be collected. A low-voltage current (0 to 30 volts with 0.5 to 1.0 amperage) is passed through the probe, stimulating ejaculation. This method is often used in bovine and ovine. The electro-ejaculator has also been used in humans with lower-body paralysis in order to obtain an ejaculate. While this method of collection is effective, it can produce lower quality semen samples when compared to the artificial vagina or digital manipulation. The electro-ejaculator should never be used in the equine due to possible tearing of the rectal tissue.
Digital manipulation is another method used for semen collection. This technique involves the physical manipulation of the penis by the collector to obtain an ejaculate. This method can be used alone with a collection container or combined with an artificial vagina. This technique is commonly used in the porcine and the canine.
The most common method for semen collection in the canine is with the use of an artificial vagina and digital manipulation. This technique involves the collector utilizing digital stimulation to encourage the dog to extend his penis and ejaculate into the artificial vagina. The index finger and thumb are placed in a u-shape behind the bulbus glandis, which helps to give the male the sensation of being “locked”. It is helpful with some animals to allow the dog to step over the collector's arm, simulating the natural tie. Depending on the individual animal, variations in pressure, friction, and movement may be necessary to obtain an ejaculate.
Regardless of which method is being used to obtain a semen sample, all must use some type of collection container. With the traditional collection methods, the collection containers (in the case of the canine, usually a plastic centrifuge tube) do not have any media placed in them prior to collection. After the semen is collected the extender will be added anywhere from a few minutes, up to one hour post-collection, depending on the protocol being used. While this method works and continues to be used today, it may not be the most efficient method.
Sperm are especially susceptible to changes in temperature. With natural service, semen is ejaculated into the warm moist environment of the female tract. However, with the traditional method of semen collection, semen is collected into a dry container where variations in temperature can be a real problem, especially if the collection room is cold. When using a dry collection tube, the outside air temperature is quickly transmitted to the semen sample. One study on mouse spermatozoa showed that sperm collected at 0° to 4° C. had significantly lower motility than that of sperm collected at 22° C. Occasionally, attempts to maintain the temperature of the collection tubes have been performed. Although attempting to maintain the temperature of the collection tube is of some help, the semen is still being shocked. The semen is coming in contact immediately with the collection container and any temperature variation can still be a problem.
Sperm are also susceptible to shifts in pH. While semen extenders do contain buffers, the semen may not come in contact with these buffers until well after collection. The semen being collected into the dry collection tubes does not allow the sperm to be buffered immediately. Because sperm are continually metabolizing, the pH will quickly change in the absence of a buffer.